The overall goal of this proposal is to uncover the mechanisms of protein trafficking utilized by the vertebrate photoreceptor to establish and maintain its unique subcellular compartmentalization. The plasma membrane of the photoreceptor consists of two major compartments with distinct protein composition and function - the outer segment plasma membrane and the inner segment plasma membrane. This separation is essential for photoreceptors to produce a reliable electrical response to light. Previous studies revealed that certain outer segment membrane proteins contain specific targeting signals to ensure they are properly sorted. However, nothing is known about the molecular and cellular mechanisms responsible for protein targeting to the plasma membrane surrounding the rest of the photoreceptor cell. In the experiments proposed in this application we will investigate the mechanisms controlling the targeting of three different proteins to the inner segment plasma membrane: the hyperpolarization-activated cyclic nucleotide-gated channel (HCN1), Na/K-ATPase and synaptophysin. We propose that the mechanism controlling the targeting of both HCN1 and Na/K-ATPase involves ankyrin-B, an essential adaptor protein within the membrane cytoskeleton. The membrane cytoskeleton lines the plasma membrane of all cells;it provides mechanical stability and regulates the protein composition of membrane subdomains. In photoreceptors, ankyrin-B decorates the entire plasma membrane outside the outer segment, which makes it an attractive candidate for serving as a master organizer of protein targeting into this domain. Aim 1 tests the hypothesis that HCN1 binds to ankyrin-B and that this interaction is required for the targeting of HCN1. Aim 2 tests the hypothesis that in photoreceptors the selective interaction of Na/K-ATPase, a known ankyrin binding protein, with ankyrin-B but not other ankyrins is a feature of the specific alpha subunit of Na/K-ATPase expressed in photoreceptors and that this selective interaction is necessary for the targeting of Na/K-ATPase. Notably, interactions with ankyrin-B are very stable, whereas some proteins are targeted to the plasma membrane transiently prior to their incorporation into organelles such as synaptic vesicles. In Aim 3 we will focus on one such synaptic vesicle resident protein, synaptophysin. We have found a novel targeting sequence within synaptophysin and will pursue this finding by identifying the protein(s) that bind to this sequence and regulate the targeting of synaptophysin. Together, these experiments will provide mechanistic insight into two targeting pathways used to drive membrane proteins specifically to the inner segment plasma membrane. PUBLIC HEALTH RELEVANCE: This work is relevant to understanding the molecular bases of blinding degenerative diseases of the retina, often characterized by disorders in photoreceptor protein trafficking or the maintenance of cellular compartmentalization. Elucidating the mechanisms controlling these processes is essential for developing therapeutic interventions.